Application of gallnut tannic acid as additive in boar production

Abstract

The invention discloses an application of gallnut tannic acid as an additive in boar production. The application of the gallnut tannic acid as the additive comprises the application of the gallnut tannic acid as a feed additive in improving the reproductive performance of adult breeding boars and the application of the gallnut tannic acid as a semen diluent additive in improving the normal-temperature preservation quality of boar semen. The feed additive can improve the reproductive performance of adult breeding boars, can significantly improve the sperm density, the total sperm number and the effective sperm number of the boars after being fed for 12 weeks, and improves the oxidation resistance of sperms, sperms and serum; the semen diluent additive can improve the normal-temperature preservation quality of the boar semen, and the sperm motility can still reach 61.20% after the boar semen is preserved for 7 days.

Description

Technical Field

[0001] The invention belongs to the technical field of functional feed for pig production and breeding, and particularly relates to application of gallnut tannic acid as an additive in boar production. Background Art

[0002] The pig industry occupies a pivotal position worldwide, especially in China, where it plays an irreplaceable role in ensuring the national meat consumption demand. Reproductive traits are important economic traits of pigs, and the reproductive performance of boars is the starting point of the pig farm production chain. Their semen quality, genetic advantages and health status directly determine the reproductive efficiency of the sow group and the growth potential of the offspring. At present, artificial insemination (AI) is an important reproductive technology for pig production, but semen preservation is restricted by many factors, among which oxidative stress is considered to be the main factor in the reduction of semen quality. Based on the redox balance theory, many researchers are paying more and more attention to the use of exogenous antioxidants to regulate the oxidative stress status of livestock and poultry to improve their production performance. Studies have shown that adding wolfberry to boar diets can significantly improve sperm motility, sperm density and total sperm count, and reduce sperm deformity rate. [1] Compared with the control group, the addition of the Chinese herbal medicine Yiyangkang to the diet significantly improved the sperm count, sperm density and motility of the boars, reduced the number of sperm abnormalities, and significantly increased the levels of reproductive hormones such as FSH, LH and T in the boars. [2] .

[0003] Tannic acid (TA) is a natural plant polyphenol compound that is widely distributed in plant-derived raw materials such as gallnut, tara, and chestnut. Gallnut is a traditional Chinese medicinal material. Its main active ingredient is gallnut tannin acid (GTA). The production in my country is extremely high and the natural active ingredient is TA, with a content of up to 70%. [3] Based on TA's antioxidant activity, metal chelation ability and film-forming properties, it has multifunctional application potential in the fields of food preservation, heavy metal adsorption, feed stabilizer and bioadhesive material. [4] Studies have found that adding an appropriate amount of TA to animal diets can significantly increase the height of ileal villi in weaned piglets, reduce intestinal permeability in weaned piglets, and improve the intestinal health of piglets. [5] The study also found that TA has strong antioxidant capacity and is a natural antioxidant that can exert antioxidant activity by scavenging ROS and relieve oxidative stress. [6,7] Supplementation with 1.5g of acacia tannin extract (0.03g / kg) and 3g of acacia tannin extract microencapsulated with palm oil (0.06g / kg) improved testicular length, sperm motility, semen volume, and sperm concentration, and reduced the percentage of abnormal sperm in Merino rams.[8] During the 96-hour storage of pig semen at room temperature, the addition of tannin-rich South African rooibos tea to the pig semen diluent improved sperm motility and protected the integrity of the sperm acrosome and plasma membrane. [9] Adding 5% crude tannin to the semen of Bali cattle and storing it at 15℃ for 14 days found that crude tannin can significantly improve sperm motility and survival rate, while reducing the abnormality rate of semen.

[10] In addition, the study also found that TA can promote the improvement of sperm motility parameters and maintenance of nuclear integrity in Brandt's vole by regulating redox homeostasis, optimizing reproductive organ development indicators, balancing serum sex hormone profiles, and regulating testicular autophagy, while also improving chromosome structural stability.

[11] Feeding lambs with grape seed tannin extract during the early stages of testicular development can upregulate the expression of genes related to antioxidant, steroidogenesis, and polyunsaturated fatty acid metabolism, enhance testicular antioxidant capacity, and contribute to testicular development and spermatogenesis.

[12] .

[0004] Therefore, the present invention provides an application of gallnut tannic acid as an additive in boar production, and it is intended to add an appropriate amount of TA to the diet of adult breeding boars to improve the antioxidant capacity of sperm, seminal plasma and serum in boar semen, thereby improving semen quality; and add an appropriate amount of TA to semen diluent to improve the antioxidant capacity of semen and sperm quality parameters during room temperature storage. Summary of the Invention

[0005] The present invention aims to provide a method for using gallnut tannic acid as an additive in boar production.

[0006] The applications of the invention include the application of the gallnut tannic acid as a feed additive in improving the reproductive performance of adult breeding boars, and the application of the gallnut tannic acid as a semen diluent additive in improving the quality of pig semen stored at room temperature.

[0007] The added amount of the gallnut tannic acid of the present invention is 0.05%-0.2% of the weight of the basic daily diet of adult boars.

[0008] Preferably, the added amount of the gallnut tannic acid of the present invention is 0.1%-0.2% by weight of the basic diet of adult boars.

[0009] Further preferably, the added amount of the gallnut tannic acid of the present invention is 0.2% by weight of the basic diet of adult boars.

[0010] The feeding frequency of the gallnut tannic acid feed additive to adult boars is twice a day, in the morning and in the afternoon.

[0011] The concentration of the gallnut tannic acid in the pig semen diluent is 3-11 μg / mL.

[0012] Preferably, the concentration of the gallnut tannic acid in the pig semen diluent of the present invention is 5-9 μg / mL.

[0013] Further preferably, the concentration of the gallnut tannic acid in the pig semen diluent of the present invention is 7 μg / mL.

[0014] The semen diluent of the present invention is Modena diluent.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention explores the effect of adding different levels of TA (0, 0.05%, 0.1%, 0.2%) to the diet of adult Duroc boars to explore the effect of adding different levels of TA on the reproductive performance of boars, thereby screening out the optimal addition amount, and examining the effect of TA on sperm, seminal plasma and serum oxidative stress indicators as well as serum testosterone levels. The results showed that the addition of 0.2% TA could significantly increase sperm density, total sperm count and effective sperm count (P<0.05); when semen was stored at room temperature for 24 hours, the addition of 0.2% TA could significantly reduce the proportion of slow-motile sperm (P<0.05); after 72 hours of storage, the addition of 0.2% TA could significantly increase the proportion of linear-motile sperm (P<0.05) and reduce the proportion of slow-motile sperm (P<0.05); the addition of 0.2% TA could significantly reduce the MDA content in sperm (P<0.05) and increase the T-AOC level and SOD activity in sperm (P<0.05); the addition of 0.2% TA could significantly reduce the MDA content in seminal plasma (P<0.05); the addition of 0.2% TA could significantly increase the GSH-PX activity in serum (P<0.05); therefore, the optimal addition amount of TA was determined to be 0.2%.

[0017] 2. This study investigated the effects of different concentrations of TA (0 μg / mL, 3 μg / mL, 5 μg / mL, 7 μg / mL, 9 μg / mL, and 11 μg / mL) added to boar semen diluents to explore the effects of different TA concentrations on the room temperature storage of boar semen and screen the appropriate concentration for addition. The results showed that the addition of 7 μg / mL of TA significantly improved sperm motility, plasma membrane, and acrosome integrity (P < 0.05), while also significantly increasing seminal serum GSH-PX and SOD activities and reducing seminal serum MDA content (P < 0.05). Therefore, the addition concentration was determined to be 7 μg / mL.

[0018] 3. The feed and semen diluent containing TA in the present invention have no adverse effects on the growth and production of pigs and semen preservation, are highly safe, and can be used as feed additives and semen diluent additives for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Effects of dietary TA supplementation on oxidative stress levels in boar sperm (A is the MDA content of each treatment group before feeding; B is the MDA content of each treatment group at the 12th week of feeding; C is the T-AOC level of each treatment group before feeding; D is the T-AOC level of each treatment group at the 12th week of feeding; E is the SOD activity of each treatment group before feeding; F is the SOD activity of each treatment group at the 12th week of feeding; G is the GSH-PX activity of each treatment group before feeding; H is the GSH-PX activity of each treatment group at the 12th week of feeding);

[0020] Figure 2 Effects of dietary TA supplementation on boar semen oxidative stress levels (A is the MDA content of each treatment group before feeding; B is the MDA content of each treatment group at the 12th week of feeding; C is the T-AOC level of each treatment group before feeding; D is the T-AOC level of each treatment group at the 12th week of feeding; E is the SOD activity of each treatment group before feeding; F is the SOD activity of each treatment group at the 12th week of feeding; G is the GSH-PX activity of each treatment group before feeding; H is the GSH-PX activity of each treatment group at the 12th week of feeding);

[0021] Figure 3 Effects of dietary TA supplementation on serum oxidative stress levels in boars (A is the MDA content of each treatment group before feeding; B is the MDA content of each treatment group at the 12th week of feeding; C is the T-AOC level of each treatment group before feeding; D is the T-AOC level of each treatment group at the 12th week of feeding; E is the SOD activity of each treatment group before feeding; F is the SOD activity of each treatment group at the 12th week of feeding; G is the GSH-PX activity of each treatment group before feeding; H is the GSH-PX activity of each treatment group at the 12th week of feeding);

[0022] Figure 4 Effects of dietary TA on serum testosterone levels in boars (A is before feeding; B is after 12 weeks of feeding);

[0023] Figure 5 Detection of the integrity of the tail of pig sperm plasma membrane (A represents sperm with damaged tail plasma membrane, and B represents sperm with intact tail plasma membrane);

[0024] Figure 6 Detection of pig sperm acrosome integrity (A is the pig sperm acrosome stained with FITC-PNA and showing green fluorescence; B is the pig sperm nucleus stained with DAPI and showing blue fluorescence; C is the Merge image);

[0025] Figure 7 Effects of adding TA to Modena diluent on sperm ROS levels;

[0026] Figure 8Effect of adding TA to Modena diluent on the MDA content of semen;

[0027] Figure 9 Effect of adding TA to Modena diluent on semen GSH-PX activity;

[0028] Figure 10 Effect of adding TA to Modena diluent on the activity of semen SOD. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described in detail below through specific embodiments.

[0030] Example 1 Application of Gallic Tannic Acid as a Feed Additive in Improving the Reproductive Performance of Adult Breeding Boars

[0031] (1) Twenty-eight healthy Duroc boars aged 14 to 16 months with similar production performance were randomly divided into four groups: one control group and three experimental groups. Each group had seven replicates, with one pig per replicate. The control group (NC) was fed a basal diet, while the three experimental groups received TA supplemented with 0.05% (experimental group I), 0.1% (experimental group II), and 0.2% (experimental group III) of the basal diet weight, respectively. The pre-feeding period was one week, and the main feeding period was 12 weeks. Feeding was done twice daily, in the morning and afternoon.

[0032] (2) Semen sample collection: Semen was collected 6 days a day during the experiment. Semen was collected strictly according to the semen collection operation requirements at weeks 0, 3, 6, 9, and 12 of the experiment, and quality testing was performed. 5 mL of semen was collected at weeks 0 and 12 in a 10 mL centrifuge tube, centrifuged at 7000 r / min for 10 min, and 3 mL of the supernatant was divided into cryopreservation tubes to obtain the semen sample. After the remaining supernatant was poured out, 5 mL of PBS was added and centrifuged at 7000 r / min for 10 min. The sample was washed repeatedly for 3 times to obtain the sperm sample. It was immediately stored in a -20°C refrigerator and then brought back to the laboratory and stored in a -80°C refrigerator for later use.

[0033] (3) Serum sample collection: At 08:00 on Sunday morning of the 0th and 12th weeks of the experiment, three fasting boars in each group were selected to collect blood from the marginal ear vein, about 10 mL. After blood coagulation, the blood was centrifuged at 3500 r / min and 4°C for 15 min to prepare serum samples. The samples were divided into 2 mL cryovials, immediately stored in a -20°C refrigerator, and then brought back to the laboratory and stored in a -80°C refrigerator for later use.

[0034] (4) Preparation of sperm homogenate: Sperm samples frozen at -80°C were thawed in a 37°C water bath, resuspended in 1 mL of normal saline, and intermittently ultrasonicated on ice (5 min, 20-30 s / time). The supernatant was collected for subsequent analysis after centrifugation at 10,000 × g at 4°C for 10 min.

[0035] (5) Perform semen quality tests, sperm tail plasma membrane and acrosome integrity tests, measure the oxidative stress levels of sperm, seminal plasma and serum, test serum testosterone content, and analyze the results.

[0036] Example 2 Application of Gallic Tannic Acid as a Semen Diluent Additive in Improving the Quality of Pig Semen Preserved at Room Temperature

[0037] (1) Preparation of TA stock solution: Accurately weigh 20 mg of TA and dissolve it in 2 mL of sterile ultrapure water to prepare a 10 mg / mL TA stock solution. Vortex and mix thoroughly. Use immediately after preparation.

[0038] (2) Preparation of Modena diluent: Accurately weigh 27.5 g of glucose, 6.9 g of trisodium citrate, 2.35 g of ethylenediaminetetraacetic acid (EDTA), 1.0 g of sodium bicarbonate, 0.75 g of potassium chloride, 2.9 g of citric acid, and 5.65 g of Tris, add 1 L of double-distilled water, place in a 2 L sterile beaker, stir to fully dissolve, filter through a 0.22 μm filter after dissolution, and use immediately.

[0039] (3) Using the prepared Modena diluent as the control group, the 10 mg / mL TA stock solution was accurately diluted into the Modena diluent according to the experimental TA concentration gradient (3, 5, 7, 9, 11 μg / mL) to prepare the experimental semen diluent, and 3 replicates were set for each concentration.

[0040] (4) Fresh semen from three healthy adult Duroc boars was collected to obtain raw semen. The raw semen was randomly divided into 6 groups and equilibrated with Modena diluent and the semen diluent for each test group in a water bath for 10 minutes. The temperature difference between the raw semen and the diluent was not more than 1°C before dilution. The sperm density after dilution was approximately 180 million / mL, and the total volume was 10 mL. The semen was placed in a clean 15 mL sterile centrifuge tube and labeled. The tube was wrapped with 16 layers of gauze and placed in a foam box at room temperature of 22-25°C to avoid light for 1-2 hours. The tube was then placed flat in a 17°C incubator and mixed up and down every 12 hours to prevent sperm sedimentation. The storage period was 7 days.

[0041] (5) Semen quality, sperm tail plasma membrane and acrosome integrity, and sperm oxidative stress level were tested on days 1, 3, 5, and 7, and the results were analyzed.

[0042] In order to verify the effectiveness of the present invention, the invention team conducted a series of experiments, as follows:

[0043] 1 Materials and Methods

[0044] 1.1 Test materials

[0045] The Duroc boars used in this study were sourced from a boar station affiliated with Guizhou Fuzhiyuan Agricultural Technology Co., Ltd. The TA used in the feeding experiment was provided by Guiyang Beilong Biotechnology Co., Ltd. (natural tannin content ≥ 40%). The TA added to the porcine semen Modena diluent was purchased from Beijing Solaibao Technology Co., Ltd. (Beijing, China, IT0710, T ≥ 99%).

[0046] 1.2 Experimental design

[0047] 1.2.1 TA feeding for adult breeding boars

[0048] Twenty-eight healthy Duroc boar pigs aged 14 to 16 months with similar production performance were randomly divided into four groups: one control group and three experimental groups, each with seven replicates and one pig per replicate. The control group (NC) was fed a basal diet, while the three experimental groups were fed diets supplemented with 0.05% (experimental group I), 0.1% (experimental group II), and 0.2% (experimental group III) TA, respectively. The pre-feeding period lasted one week, and the main feeding period lasted 12 weeks.

[0049] 1.2.2 Preservation of boar semen by adding TA diluent

[0050] The experiment consisted of one control group and five treatment groups. Using the prepared Modena diluent as the control group, 10 mg / mL TA stock solution was precisely diluted into the Modena diluent according to the experimental TA concentration gradient (3, 5, 7, 9, and 11 μg / mL) to prepare the experimental semen diluents, with three replicates for each concentration. The raw semen and Modena diluent were equilibrated in a water bath for 10 minutes, ensuring a temperature difference of no more than 1°C before dilution to obtain fresh mixed semen. The diluted sperm density was approximately 180 million / mL, and the total volume was 10 mL. The semen was placed in a clean, sterile 15 mL centrifuge tube, labeled, wrapped with 16 layers of gauze, and placed in a foam box at 22-25°C, protected from light, for 1-2 hours. The tube was then placed flat in a 17°C incubator, mixing thoroughly every 12 hours to prevent sperm sedimentation.

[0051] 1.3 Preparation of main solutions

[0052] Preparation of TA stock solution: Accurately weigh 20 mg of TA and dissolve it in 2 mL of sterile ultrapure water to prepare a 10 mg / mL TA stock solution. Vortex and mix thoroughly. Use immediately after preparation.

[0053] Preparation of Modena diluent: Accurately weigh 27.5 g of glucose, 6.9 g of trisodium citrate, 2.35 g of ethylenediaminetetraacetic acid (EDTA), 1.0 g of sodium bicarbonate, 0.75 g of potassium chloride, 2.9 g of citric acid, and 5.65 g of Tris. Add 1 L of double-distilled water and place in a 2 L sterile beaker, stirring to fully dissolve. After dissolution, filter through a 0.22 μm filter and use immediately.

[0054] 1.4 Semen collection and processing

[0055] Semen samples were collected 6 times daily during the trial. Semen was collected at weeks 0, 3, 6, 9, and 12 according to strict semen collection procedures and quality tested. 5 mL of semen was collected at weeks 0 and 12 in 10 mL centrifuge tubes and centrifuged at 7000 rpm for 10 minutes. 3 mL of the supernatant was aliquoted into cryopreservation tubes to obtain the semen serum samples. After decanting the remaining supernatant, 5 mL of PBS was added and centrifuged at 7000 rpm for 10 minutes. Repeat this process three times to obtain the semen samples, which were immediately stored at -20°C and then brought back to the laboratory and stored at -80°C until further use.

[0056] Serum samples: At 8:00 am on Sunday morning of the 0th and 12th weeks of the experiment, three fasting boars in each group were selected for blood collection from the marginal ear vein (about 10 mL). After blood coagulation, the samples were centrifuged at 3500 rpm and 4°C for 15 min to prepare serum samples. The samples were divided into 2 mL cryovials, immediately stored in a -20°C refrigerator, and then brought back to the laboratory and stored in a -80°C refrigerator for later use.

[0057] Preparation of sperm homogenate: Sperm samples frozen at -80°C were thawed in a 37°C water bath, resuspended in 1 mL of normal saline, and intermittently ultrasonicated on ice (5 min, 20-30 s / time). The supernatant was collected for subsequent analysis after centrifugation at 10,000 × g at 4°C for 10 min.

[0058] 1.5 Semen quality testing

[0059] Ejaculate volume: The semen mass was accurately weighed using an electronic analytical balance (volume was converted based on a density of 1 g / mL).

[0060] Color and smell: Visually determine the milky white color of semen under natural light conditions, and smell to identify the characteristic fishy smell and eliminate any odor of corruption.

[0061] Sperm motility, density (total sperm count, effective sperm count), morphology (deformity rate), and kinematic parameters were tested using the CASA system (automatic sperm quality analyzer, Nanning Songjing Tianlun Biotechnology Co., Ltd., Nanning, China) in strict accordance with the operational requirements of the boar station for semen quality analysis.

[0062] 1.6 Detection of sperm tail plasma membrane and acrosome integrity

[0063] In this study, the sperm viability detection kit (hypotonic swelling method, Beijing Leigeng Biotechnology Co., Ltd., Beijing, China) and FITC-PNA / DAPI fluorescence staining method (Sigma, USA / Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China) were used to detect the integrity of the plasma membrane and acrosome of pig sperm tails at days 1, 3, 5, and 7. The specific operation was referred to the methods in previous literature.

[13] .

[0064] 1.7 Determination of oxidative stress levels in sperm, seminal plasma, and serum

[0065] The MDA content, T-AOC, SOD activity, GSH-PX activity, and ROS level of sperm, seminal plasma, and serum were determined strictly according to the instructions of the detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

[0066] 1.8 Serum testosterone level detection

[0067] The testosterone content in serum was detected strictly according to the instructions of the testosterone detection kit (Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China).

[0068] 1.9 Data Statistics and Analysis

[0069] All data were analyzed for normality and homogeneity of variance using IBM SPSS Statistics 27. One-way ANOVA was performed, and Duncan's method was used for multiple comparisons between treatment groups to determine significant differences. Data are expressed as mean ± standard deviation (mean ± SD), with a P value < 0.05 considered significant. Furthermore, two-way ANOVA was used to analyze the effects of time and treatment. GraphPad Prism 9.5 software was used to plot and annotate the differences.

[0070] 2 Results and Analysis

[0071] 2.1 Effect of TA on semen quality of adult breeding boars

[0072] 2.1.1 Effect of dietary TA supplementation on boar semen quality

[0073] Table 1 shows that there was no interactive effect between feeding time and dietary treatment on conventional boar semen quality parameters (P>0.05). Feeding time significantly affected semen yield, sperm deformity rate, sperm density, total sperm count, and viable sperm count (P<0.05). Although semen yield increased throughout the experimental period, dietary treatment had no significant effect on semen yield (P>0.05). Differences in sperm motility began to emerge between groups at week 9. At week 12, sperm motility in Groups I and II was significantly higher than that in the NC group (P<0.05), while there was no significant difference in Group III (P>0.05). Furthermore, the sperm deformity rate in Group I was significantly lower than that in the other three groups (P<0.05). At week 12, sperm density and total sperm count in Group III were significantly higher than those in the NC group (P<0.05), while there were no significant differences between Groups I and II and the NC group (P>0.05). However, at week 12, the effective sperm counts in each TA group were significantly higher than those in the NC group (P<0.05).

[0074] Table 1 Effects of dietary TA on boar semen quality

[0075]

[0076]

[0077]

[0078] 2.1.2 Effects of dietary TA supplementation on boar sperm motility parameters

[0079] As shown in Table 2, there was no interactive effect between feeding time and dietary treatment on boar sperm motility parameters (P>0.05). Feeding time did not significantly affect sperm motility parameters (linear, slow, and stationary sperm ratios) in any of the groups (P>0.05). Furthermore, dietary treatment had no significant effect on the linear and slow sperm ratios (P>0.05). At week 9, the stationary sperm ratio in Group I was significantly lower than that in the other three groups (P<0.05). At week 12, the stationary sperm ratios in Groups I and II were significantly lower than those in Groups NC and III (P<0.05), but there was no significant difference between Group III and the NC group (P>0.05).

[0080] Table 2 Effects of dietary GTA supplementation on sperm kinematic parameters in fresh boar semen

[0081]

[0082]

[0083]

[0084] 2.1.3 Effect of dietary TA supplementation on the quality of boar semen stored at room temperature

[0085] Semen was collected from pigs fed for 12 weeks and stored at room temperature. As shown in Table 3, during room-temperature storage, no statistically significant differences were observed between the TA and NC groups in sperm motility, deformity rate, and the proportion of sperm motility in situ (P>0.05). At 24 hours of semen storage, the proportion of slow-motile sperm in Groups II and III was significantly lower than that in Groups NC and I (P<0.05). By 72 hours of storage, the proportion of linearly motile sperm in Group III was significantly higher than that in Group NC (P<0.05). Furthermore, the proportion of slow-motile sperm in all TA groups was significantly lower than that in the NC group (P<0.05), with the greatest reduction in Group III.

[0086] Table 3 Effects of dietary GTA supplementation on sperm motility and kinematic parameters of boar semen stored at room temperature

[0087]

[0088]

[0089] 2.1.4 Effects of dietary TA supplementation on boar sperm, seminal plasma, serum oxidative stress, and testosterone levels

[0090] Depend on Figure 1 At week 12, the NC group had significantly higher sperm MDA levels than groups I and III (P < 0.05), but no significant difference from group II (P > 0.05). Furthermore, sperm T-AOC levels and SOD activity in groups I and III were significantly higher than those in the NC group (P < 0.05). Sperm GSH-PX activity did not show significant changes among the groups throughout the trial.

[0091] Depend on Figure 2 Results showed that the seminal serum MDA levels in the TA group were higher than those in the NC group before feeding, and significantly lower than those in the NC group at week 12 (P < 0.05). At week 12, the seminal serum T-AOC level in group I was significantly higher than that in the NC group (P < 0.05). Furthermore, the seminal serum SOD and GSH-PX activities in groups I and II were significantly higher than those in the NC group (P < 0.05).

[0092] Depend on Figure 3 As the experiment progressed, serum MDA levels in groups I and II continued to decrease, reaching significantly lower levels than those in groups NC and III at week 12 (P<0.05). Furthermore, T-AOC and SOD activities in groups I and II were significantly higher than those in groups NC and III, and GSH-PX activities in all TA groups were significantly higher than those in the NC group (P<0.05). There was no significant difference in T-AOC levels between groups III and NC (P>0.05).

[0093] Depend on Figure 4 It can be seen that before feeding, there were differences in serum testosterone levels among the groups due to individual differences, but after feeding for 12 weeks, the testosterone levels of each TA group remained unchanged compared with before feeding.

[0094] 2.2 Fresh Semen Quality Analysis

[0095] The fresh mixed semen used in the experiment was derived from semen of three qualified Duroc boars. All sperm indicators of the fresh boar semen met the test requirements and were of good quality (Table 4).

[0096] Table 4 Boar Semen Quality

[0097]

[0098] 2.2.1 Effect of adding TA to Modena diluent on sperm motility

[0099] As shown in Table 5, there was no significant difference in sperm motility between the treatment groups and the control group on days 1 and 3 of room-temperature storage. On days 5 and 7 of room-temperature storage, sperm motility in the 7 μg / mL TA and 9 μg / mL TA groups was higher than that in the control group (P < 0.05), and the 7 μg / mL TA group was significantly higher than that in all other treatment groups except the 9 μg / mL group.

[0100] Table 5 Effect of adding TA to Modena diluent on sperm motility (%)

[0101]

[0102]

[0103] Note: Data in the same column with different lowercase letters indicate significant differences (P<0.05), while data with the same or no lowercase letters indicate no significant differences (P>0.05).

[0104] Same as the table below.

[0105] 2.2.2 Effect of adding TA to Modena diluent on sperm motility

[0106] Table 6 shows that on the first day of semen storage, sperm motility in the treated groups was not significantly different from that in the control group, but all treated groups were higher than the control group. With increasing storage time, sperm motility in all groups showed a decreasing trend. On days 3, 5, and 7 of room-temperature storage, sperm motility in the 7 μg / mL TA group was higher than in the 3, 5, and 11 μg / mL TA groups and the control group (P < 0.05), and remained above 60% on day 7.

[0107] Table 6 Effect of adding TA to Modena diluent on sperm motility (%)

[0108]

[0109] 2.2.3 Effect of TA addition in Modena diluent on the integrity of the sperm tail plasma membrane

[0110] The integrity of the pig sperm plasma membrane tail is detected Figure 5 As shown in Table 7, the integrity of the sperm tail plasma membrane decreased with the extension of storage time, and the integrity of the sperm tail plasma membrane in each treatment group was higher than that in the control group. On the third day of room temperature storage, the integrity of the sperm tail plasma membrane in the 7μg / mL TA group was significantly higher than that in the control group; on the fifth day, the integrity of the sperm tail plasma membrane in the 5μg / mL TA group, the 7μg / mL TA group, and the 9μg / mL TA group was higher than that in the control group (P<0.05); on the seventh day, the integrity of the sperm tail plasma membrane in the 7μg / mL TA group and the 9μg / mL TA group was significantly higher than that in the control group (P<0.05).

[0111] Table 7 Effect of adding TA to Modena diluent on the integrity of the plasma membrane of sperm tail (%)

[0112]

[0113] 2.2.4 Effect of adding TA to Modena diluent on sperm acrosome integrity

[0114] The integrity of the acrosome of pig sperm is detected Figure 6 As shown in Table 8, on the first day of room temperature storage, there was no significant difference in the sperm acrosome integrity rate between the treatment groups and the control group. On the third day, the sperm acrosome integrity rates in the 5 μg / mL TA group, the 7 μg / mL TA group, and the 9 μg / mL TA group were higher than those in the control group (P < 0.05). On the fifth and seventh days, the sperm acrosome integrity rates in the 7 μg / mL TA group and the 9 μg / mL TA group were higher than those in the control group (P < 0.05). There were no significant differences between the other treatment groups and the control group.

[0115] Table 8 Effect of adding TA to Modena diluent on sperm acrosome integrity (%)

[0116]

[0117] 2.2.5 Effect of adding TA to Modena diluent on sperm ROS levels

[0118] As the storage time increases, the ROS level in pig sperm gradually increases. Figure 7It can be seen that on the first day of storage, the ROS levels of each treatment group were lower than those of the control group (P<0.05); on the third day, there was no significant difference in the ROS levels between the TA groups and the control group, but except for the 11μg / mL TA group, the ROS levels of the other treatment groups were lower than those of the control group; on the fifth and seventh days, the ROS levels of the 5μg / mL TA group, the 7μg / mL TA group, the 9μg / mL TA group, and the 11μg / mL TA group were lower than those of the control group (P<0.05).

[0119] 2.2.6 Effect of adding TA to Modena diluent on semen MDA content

[0120] Depend on Figure 8 On the first day of room temperature storage, MDA levels in the treatment groups, except for the 3 μg / mL TA group, showed no significant difference compared to the control group. On the third day, MDA levels in the 3 μg / mL TA, 7 μg / mL TA, and 11 μg / mL TA groups were lower than those in the control group (P < 0.05). On the fifth and seventh days, MDA levels in all treatment groups were lower than those in the control group (P < 0.05), with the 7 μg / mL TA group showing the lowest MDA level.

[0121] 2.2.7 Effect of TA Addition in Modena Diluent on Semen GSH-Px Activity

[0122] Depend on Figure 9 During the entire process of room temperature storage, GSH-Px activities were higher in the TA groups than in the control group on days 1, 3, 5, and 7, except for the 11 μg / mL TA group on day 3 (P < 0.05). Except for day 3, GSH-Px activities in the 7 μg / mL TA group were higher than those in the other treatment groups at all other time points (P < 0.05).

[0123] 2.2.8 Effect of adding TA to Modena diluent on semen SOD activity

[0124] Depend on Figure 10 The trend of SOD activity in semen was similar to that of GSH-Px. With increasing TA concentration, SOD activity initially increased and then decreased. Throughout the storage process, SOD activity in the TA group was higher than that in the control group (P < 0.05). Specifically, SOD activity in the 7 μg / mL TA group was higher than that in the other treatment groups starting on day 3, indicating that TA addition increased SOD activity in pig semen in a dose-dependent manner and that 7 μg / mL TA could effectively maintain SOD activity in semen.

[0125] Although the present invention has been described in detail above using general explanations, specific embodiments, and experiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

[0126] References

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[0139] Zhang Feng, Han Shichang, Zhang Nian, et al. Effect of resveratrol on the storage of goat semen at room temperature[J]. China Animal Husbandry Journal, 2023, 59(09): 250-253.

Claims

1. An application of gallnut tannic acid as an additive in boar production, characterized in that: The applications include the use of gallnut tannic acid as a feed additive in improving the reproductive performance of adult breeding boars, and the use of gallnut tannic acid as a semen diluent additive in improving the quality of pig semen stored at room temperature.

2. The use of gallnut tannic acid as an additive in boar production according to claim 1, characterized in that: The gallnut tannic acid is added in an amount of 0.05% to 0.2% of the weight of the basic daily diet of adult boars when used as a feed additive.

3. The use of gallnut tannic acid as an additive in boar production according to claim 2, characterized in that: The gallnut tannic acid is added in an amount of 0.1% to 0.2% of the weight of the basic daily diet of adult boars when used as a feed additive.

4. The use of gallnut tannic acid as an additive in boar production according to claim 3, characterized in that: The gallnut tannic acid is added in an amount of 0.2% of the weight of the basic daily diet of adult boars when used as a feed additive.

5. The use of gallnut tannic acid as an additive in boar production according to claim 1, characterized in that: The feeding frequency of the gallnut tannic acid feed additive to adult boars is twice a day, in the morning and in the afternoon.

6. The use of gallnut tannic acid as an additive in boar production according to claim 1, characterized in that: The concentration of the gallnut tannic acid as a semen diluent additive in the pig semen diluent is 3-11 μg / mL.

7. The use of gallnut tannic acid as an additive in boar production according to claim 6, characterized in that: The concentration of the gallnut tannic acid as a semen diluent additive in the pig semen diluent is 5-9 μg / mL.

8. The use of gallnut tannic acid as an additive in boar production according to claim 7, characterized in that: The concentration of the gallnut tannic acid as a semen diluent additive in the pig semen diluent is 7 μg / mL.

9. The use of gallnut tannic acid as an additive in boar production according to claim 1, characterized in that: The semen diluent is Modena diluent.

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